Successful delivery of large-size CRISPR/Cas9 vectors in hard-to-transfect human cells using small plasmids

With the rise of new powerful genome engineering technologies, such as CRISPR/Cas9, cell models can be engineered effectively to accelerate basic and disease research. The most critical step in this procedure is the efficient delivery of foreign nucleic acids into cells by cellular transfection. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9–19 kb), they result in low transfection efficiency and cell viability, and thus subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. At its core, the technique is based on adding exogenous small plasmids of a defined size to the transfection mixture.